Voltage-gated sodium channels are centrally important to vertebrate nerve-muscle physiology. They propagate nerve and muscle action potentials in excitable tissue. Subtle structural variation across channel isoforms likely plays a role is tissue specific patterns of excitability and function. Additionally, these channels are targets of a wide array of pharmacological agents. Understanding natural variation in sodium channel isoforms as well as functional constraints on channel evolution will help eluciidate the role structural variation in channel isoforms plays in tissue specific patterns of excitability. In addition, this understanding will aid the development of drugs and/or channel probes that may help diagnose and treat diseases associated with channelopathies. Research proposed here will examine natural structural variation in the pore region of the sodium channel. Selection towards TTX resistance in TTX bearing salamanders should drive TTX senstive channels to become TTX insensitive. However the outer pore (where TTX binds) is also critical for determining the ion selectivity and conductance of the channel. As such, functional constraints may limit the ability of salamander channel isoforms. Examining the outcome of this natural experiment will inform our understanding of vertebrate ion channel structure function relationships. This work will include training in molecular biology, neurophysiology, and molecular evolution and will explore the molecular and physiological basis of TTX resistance in TTX bearing salamanders. RT-PCR and other cloning tools will be used to sequence multiple channel genes from multiple salamander species. Tetrodotoxin resistance of salamander tissues will be characterized using simple electrophysiology as well as whole-cell patch clamp of mutant channels expressed in a heterologous expression system. Channel sequence data will be analyzed for evidence of positive selection and those results will be used to investigate functional constraints on the molecular structure of vertebrate voltage-gated sodium channels. Relevance to human health-Because channels are highly conserved across vertebrates work here will be directly relevant to structure-function questions in human voltage-gated sodium channels and will extend our understanding of the pore region of these channels. This research may also aid in the development of new pharmacological tools for the evaluation and treatment of diseases associated with channelopathies.